ABSTRACT An unanswered question of large public health importance for women living in settings with high HIV burden is whether the use of any hormonal contraceptive method, and injectable depot medroxyprogesterone acetate (DMPA) in particular, increases HIV risk. The objective of this proposal is to conduct biologic studies of the potential mechanisms by which hormonal contraceptives may render women more susceptible to HIV infection. A rigorous clinical trial, ?ECHO,? to definitively address the question of HIV acquisition risk with the use of contraceptives has just enrolled the first participants. In the trial, women are randomly assigned to use injectable DMPA, the levonorgestrel (LNG) contraceptive implant, or the non-hormonal T-380 copper intrauterine contraceptive device (IUD), equally effective contraceptive methods, positioning the trial to overcome many of the challenges that have plagued prior observational studies. Through the proposed work, we will enroll a comparable group of women using no contraception to serve as an additional control group. The trial does not include provisions to collect genital samples or conduct mechanistic studies. This proposal seizes an unprecedented opportunity to conduct studies nested within ECHO to test the most promising hypotheses about mechanisms by which hormonal contraceptives may increase HIV risk, including that hormonal contraceptives elicit: 1) increases in vaginal phylotypes associated with HIV risk, 2) increases in inflammation leading to HIV target cell recruitment or activation, 3) increases in target cell homing pathways, and/or 4) disruptions in mucosal barrier integrity. We will use state-of-the-art laboratory techniques ? including qPCR, 16S rRNA sequencing, flow cytometry, targeted and discovery mass spectrometry-based proteomics, and SmartSeq ? to achieve the study aims. The specific aims are to: 1) identify key vaginal phylotypes altered via initiation of hormonal contraceptives (DMPA versus copper IUD, DMPA versus no contraception, DMPA versus LNG implant, etc.) that mediate heightened susceptibility to HIV infection; 2) examine whether initiation of hormonal contraceptives elicits increased genital inflammation and HIV target cells and whether this correlates with increased HIV risk; and 3) determine whether initiation of hormonal contraceptives elicits increases in target cell homing pathways, as measured by the vaginal transcriptome, and/or decreases in epithelial integrity, as measured by secreted proteome, and whether these changes correlate with HIV infection. Finally, these large mucosal databases will be integrated with data-driven computational modeling to generate testable hypotheses about pathways leading to HIV infection. This study will identify mechanisms through which hormonal contraceptives alter genital mucosa, and may identify molecular and cellular biomarkers of increased mucosal HIV susceptibility. Results from this study will be pertinent for contraceptive recommendations for women with HIV risk.